This proposal seeks to quantify the expression of thousands of mRNAs using microarray technologies in order to determine the range and magnitude of variation in mRNA expression among individuals and populations and to apply evolutionary analyses in order to determine how much of this variation is either neutral or, alternatively, biologically important. Based on our knowledge of genetic variation, many of the changes in mRNA expression will represent random neutral genetic differences, differences that do not produce biologically important phenotypic changes. The application of evolutionary analyses can identify these random variations in mRNA expression. Alternatively, some of these variations in mRNA expression have evolved by natural selection and thus are biologically important. Among the teleost fish Fundulus, populations in four different species have independently evolved adaptive differences in glycolytic enzyme expression and cardiac metabolism (Pierce. V. A. and D. L. Crawford, 1997, Science 275:256-259). These four species provide replicate populations to inquire whether similar changes in gene expression have evolved. This proposed research seeks to take advantage of the differences among populations and species of Fundulus to identify which variations in mRNA expression are adaptively important and relate these adaptive gene complexes to the variation in cardiac metabolism. Quantifying genome-wide variation in mRNA expression using vertebrate species that have evolved differences in metabolism and gene expression addresses basic questions concerning the importance of all the enzymes in effecting a change in metabolic flux. This evolutionary approach provides the means to distinguish random variation from adaptively important variation and will lead to the discovery of patterns of mRNA expression that affect cardiac physiology.